New materials and methods of synthesis are emerging as significant areas of research. They have applications in the fields of biotechnology, medicine, pharmaceuticals, medical devices, polymers, etc. The ring-opening metathesis polymerization (ROMP) method has emerged as a powerful synthetic method for the creation of such useful materials. Many examples in which ROMP has been used to generate functionalized materials have focused on the incorporation of pendant functionality into the monomers, thereby forming a multivalent array. As used herein, a multivalent array refers to a polymer (random or block of varying lengths, including shorter oligomers) having pendant functional groups that impart various properties to the polymer. Such multivalent arrays are also often referred to as multivalent ligands, multivalent displays, multidentate arrays, multidentate ligands, or multidentate displays.
Such multivalent arrays are particularly useful in the medical and biotechnology areas. For example, the binding of cell surface receptors to particular epitopes of multivalent arrays can trigger a wide variety of biological responses. Such multivalent binding events have unique consequences that are dramatically different than those elicited by monovalent interactions. For instance, signaling through the epidermal growth factor is promoted by the binding of divalent ligands, which apparently promote dimerization of the transmembrane receptor, yet monovalent ligands also bind the receptor but produce no signal. In addition, multivalent arrays have been shown to induce the release of a cell surface protein, suggesting a new mechanism for controlling protein display. In protein-carbohydrate recognition processes, multivalent saccharide-substituted arrays can exhibit increased avidity, specificity, and unique inhibitory potencies under dynamic conditions of shear flow. Thus, the ability to synthesize defined, multivalent arrays of biologically relevant binding epitopes provides a means for exploring and manipulating physiologically significant processes.
Because they can span large distances, linear multivalent arrays of varying length and epitope density are particularly useful for probing structure-function relationships in biological systems. Chemical and chemoenzymatic routes have been developed for the generation of di- and trivalent ligands, dendrimers, and high molecular weight polymers, but well defined, linear oligomers have proven more difficult to synthesize. Thus, what is needed is a general strategy to create diverse arrays of different multivalent materials of varying length.
One way in which this could be done is through the use of ROMP technology. ROMP has been used to generate defined, biologically active polymers (Gibson et al., Chem. Commun., 1095-1096 (1997); Biagini et al., Chem. Commun., 1097-1098 (1997); Biagini et al., Polymer, 39, 1007-1014 (1998); and Kiessling et al., Topics in Organometallic Chemistry, 1, 199-231 (1998)) with potent and unique activities that range from inhibiting protein-carbohydrate recognition events to promoting the proteolytic release of cell surface proteins (Mortell et al., J. Am. Chem. Soc., 118, 2297-2298 (1996); Mortell et al., J. Am. Chem. Soc., 116, 12053-12054 (1994); Kanai et al., J. Am. Chem. Soc., 119, 9931-9932 (1997)); Kingsbury et al., J. Am. Chem. Soc., 121, 791-799 (1999); Schrock et al., J. Am. Chem. Soc., 112, 3875-3886 (1990); Gordon et al., Nature, 392, 30-31 (1998); and Sanders et al., J. Biol. Chem., 274, 5271-5278 (1999)). The assembly of multivalent materials by ROMP has several advantages over classical methods for generation of multivalent displays. Specifically, ROMP can be performed under living polymerization conditions, and if the rate of initiation is faster than that of propagation, varying the monomer to initiator ratio (M:I) can generate materials of defined length (Ivin, Olefin Metathesis and metathesis polymerization; Academic Press: San Diego, 1997). This approach has been successfully applied with the Grubb""s ruthenium metal carbene catalyst ([(Cy)3P]2Cl2Ruxe2x95x90CHPh) to generate materials with narrow polydispersities, indicating that the resulting substances are fairly homogeneous (Dias et al., J. Am. Chem. Soc., 119, 3887-3897 (1997); Lynn et al., J. Am. Chem. Soc., 118, 784-790 (1996)). In contrast to anionic and cationic polymerization catalysts, ruthenium metal carbene initiators are tolerant of a wide range of functional groups.
There are, however, inherent disadvantages in the use of standard approaches that rely on ROMP to assemble biologically active materials. For example, the desired pendant functionality is incorporated into the monomers. Thus, a new functionalized cyclic olefin monomer, typically a functionalized bicyclic monomer, must be synthesized for each new polymer class to be produced. Also, the physical properties of each monomer, such as its solubility and the electron density and strain of the cyclic olefin, result in different rates of initiation, propagation, and non-productive termination of the reaction (Kanai et al., J. Am. Chem. Soc., 119, 9931-9932 (1997)). In addition, purification of the desired products can be complicated depending on the structure of the monomer used.
Expedient, large-scale syntheses of multivalent arrays are hindered by these technical complications. Thus, what is needed is a general method for synthesizing multivalent arrays that addresses one or more of these issues. Ultimately, both large-scale production and the generation of libraries of oligomers would be facilitated by such a method.
The present invention provides methods for synthesizing multivalent arrays, such as functionalized polymers (herein, included within this term are relatively short oligomers). Significantly, the methods of the present invention can provide access to a wider range of materials with significant functions. For example, they can be used to generate libraries of oligomeric substances that differ in appended functionality as well as in length. Significantly, the methods of the present invention provide the ability to control the number and type of pendant functional groups. Such design control is important for elucidating structure/function relationships in biological systems, for example. The methods of the present invention can be used to produce random copolymers (i.e., polymers derived from two or more different monomers). In addition, block copolymers can be generated in which some blocks are held invariant while others are diversified through the method of the present invention. The blocks can vary in the backbone and/or the pendant functional groups.
In one embodiment, the present invention provides a method of preparing a multivalent array. The method includes: polymerizing at least one monomer comprising at least one polymerizable group and at least one latent reactive group in the presence of a metal carbene catalyst to form a polymer template comprising at least one latent reactive group; and combining the polymer template with at least one functionalizing reagent comprising at least one reactive group under conditions effective to react the latent reactive group of the polymer template with the reactive group of the functionalizing reagent to form a multivalent array. The monomer can optionally include functional groups nonreactive with the reactive group of the functionalizing reagent (herein, referred to as prefunctionalized monomers). In one embodiment, the latent reactive group of the monomer includes a nucleophilic group and the reactive group of the functionalizing reagent includes an electrophilic group. In another embodiment, the latent reactive group of the monomer includes an electrophilic group and the reactive group of the functionalizing reagent includes a nucleophilic group. In a particularly preferred embodiment, the electrophilic group is an activated ester group and the nucleophilic group is a primary amine group.
The polymer template, and hence, the multivalent array, can be a block copolymer or a random copolymer. A block copolymer is formed by the method described above wherein polymerizing at least one monomer comprises sequentially polymerizing two or more different monomers in the presence of a metal carbene catalyst to form a polymer template comprising alternating blocks of the different monomers. Alternatively, a random copolymer is formed by the method described above wherein polymerizing at least one monomer comprises simultaneously polymerizing two or more different monomers. Each different monomer can include a different latent reactive group for subsequent attachment of pendant functional groups. Such pendant functional groups can be derived from functionalizing reagents that react with the latent reactive group of the polymer template comprising a carbohydrate or a peptide.
The present invention also provides polymer templates and kits that include at least one polymer template. The kits also include instruction means for using a functionalizing reagent to attach a pendant functional group to the polymer template. The kits can also includes at least one functionalizing reagent and/or at least one capping agent.